Pneumocystis has risen to the forefront of lethal, opportunistic lung infections as a result of its close association with the HIV epidemic, yet this atypical fungal pathogen is becoming increasingly associated with pharmacologic-driven immunosuppression as well as patients with chronic lung diseases such as COPD. Loss of CD4+ T cells is a known predisposing factor to Pneumocystis pneumonia, yet how CD4+ T cells specifically control Pneumocystis infection is not known. The lack of the ability to culture Pneumocystis in vitro coupled with high species specificity has limited investigations into human CD4 T cell mediated responses to Pneumocystis. T helper immunity to Pneumocystis is complicated, in that mice deficient in the T helper type 1 (Th1) signature cytokine IFN-? or the Th2 signature cytokine IL-4 are not more susceptible to infection than wild-type animals. Although some data points to Th2 responses being protective, in many experimental models, evidence mostly supports a role for Th1 responses in mediating protective immunity to P. murina. Delayed (higher burden at 2-3 weeks compared to wild-type), but intact (no differences in burden at 4-5 weeks compared to wild-type) clearance of Pneumocystis has been observed in CD4-competent IFN-?R deficient mice, IL-12p35 deficient mice and IL-23p19 deficient mice. One commonality between these cytokines is that IFN-?, IL-12p35 and IL-23p19 each activate the pro-Th1 transcription factor Signal Transducer and Activator of Transcription 4 (STAT4). Therefore, to more formally address the role of T helper development in P. murina host defense, we focused on specific transcription factors that drive Th1 and Th2 responses. In preliminary studies, we show that CD4-competent mice deficient in STAT4, but not the pro-Th2 transcription factor STAT6, are unable to clear P. murina from the lungs 4 weeks post-challenge. At 2 weeks post-challenge, when P. murina burden was similar between all groups, STAT4-/- mice demonstrated reduced lung levels of IL-1?, IL- 1?, CXCL1, CCL2 and CCL5 as well as reductions in total CD3+, CD3+/CD4+ and MHC II+ cells in lung digests. Upon anti-CD3 stimulation, STAT4-/- CD4+ T cells displayed impaired production of IL-2 and IFN-?. As P. murina lung clearance in IFN-?R, IL-12p35 and IL-23p19 deficient mice was impaired early in infection, rather than late (4 weeks) as observed in STAT4-/- mice, these results suggest that an additional mediator may synergize with IFN-?, IL-12p35 or IL-23p19 to maximize STAT4 activation and the subsequent anti- Pneumocystis immune response. To this end, we show that the STAT4 activator IL-18, as well as the Th1 promoting cytokine IL-27, are induced in the lungs during P. murina infection. Surprisingly, we found that mice deficient in the Th1-associated transcription factors T-bet or STAT1 did not display impaired clearance of P. murina 4 weeks post-challenge, indicating a strict requirement for STAT4 in lung clearance of P. murina. Therefore, the focus of this R21 Exploratory/Developmental Research Grant is to define mechanisms of STAT4 induction and mechanisms of STAT4-mediated adaptive immune defense pathways that mediate lung clearance of P. murina. Our central hypothesis is that signaling through STAT4 mediates protective lung immunity against Pneumocystis. To address this hypothesis, we have proposed the following two independent, interrelated Aims: (1) Establish roles for IL-18 and IL-27 in STAT4 signaling and host defense against P. murina lung infection and (2) Elucidate the STAT4-dependent CD4 T cell phenotype during Pneumocystis lung infection. PUBLIC HEALTH RELEVANCE: Our data has uncovered a new understanding of the lung immune response to Pneumocystis, a significant cause of pneumonia in immunosuppressed individuals. We have discovered that CD4+ T cells require the transcription factor STAT4 to mediate host defense against Pneumocystis. Studies in this proposal will define mechanisms of STAT4 induction and mechanisms of STAT4-mediated adaptive immune defense pathways that mediate lung clearance of Pneumocystis.